This invention generally relates to microparticles with unique characteristics detectable by instruments and methods for use in the measurement of analytes in fluids.
Fluorescent polymeric particles have often found utility as markers and indicators in various biomedical assays. Fluorescent particles are usually obtained by embedding or diffusing a fluorescent dye according to the technique originally described by L. B. Bangs (Uniform Latex Particles; Seragen Diagnostics Inc. 1984, p. 40). Other methods are known in the art to stain particles with fluorescent dyes. The microparticles can then be analyzed manually or by other methods known in the art, but preferably using an automated technique, e.g., flow cytometry, such as disclosed in U.S. Pat. No. 4,665,024 issued to Mansour, et al.
The versatility of these particles can be enhanced by the incorporation in a single particle of a plurality of dyes, each dye having unique spectral characteristics. One skilled in the art would recognize that two or more dyes of varying proportions could be used to increase the permutation number of unique combinations of dyes on a single particle. While simple absorption of a single dye into a particle has proven adequate for most purposes, several problems arise when more than one dye is absorbed into a particle.
First, the close proximity of embedded dye molecules gives rise to significant amounts of fluorescent energy transfer. This energy transfer leads to fluorescent emissions that are inconsistent with relative dye concentrations and their original emission patterns.
A second problem arises when the dye substances used have differing solubilities in the solvent used to incorporate the dye in the particles. Since all dyes must be absorbed simultaneously, possible dye ratios are restricted by solvent properties.
A third problem that has been encountered when multiple dyes are embedded in microparticles is the change in dye spectra. Specifically, it has been noted that, when the particle is composed of crosslinked polystyrene, a significant broadening of the fluorescent emission peak occurs. This can result in an overlapping of the spectra of neighboring dyes.
One method that may circumvent these problems is to chemically couple each dye substance to the surface of the particle. This approach is, for example, disclosed in U.S. Pat. Nos. 5,194,300 Cheung and 4,774,189 Schwartz, whereby one or several fluorescent dyes are covalently bound to the surface of particles. This, however, leaves the dye molecule exposed to the environment, which can hasten decomposition by oxidation or other chemical attack. Additionally, a large number of surface binding sites would be occupied by dye and would be unavailable for the conjugation of analytical reactant molecules necessary to perform the assays.
Hence, it is desirable to have multicolored fluorescent particles, which avoid the above problems. This invention minimizes or eliminates these complications while maintaining the versatility of multi-dye particles.
Masson et al., disclose in U.S. Pat. No. 4,279,617, latex particles of relatively large diameter (e.g. 0.79 xcexcm) coated with an analytical reactant, e.g., allergen, either by simple adsorption or by covalent coupling with cyanogen bromide or hydroxylated latex. A sample of human serum from a person suspected to have an allergic reaction, is mixed with a suspension of these particles. The mixture is incubated and latex particles of a relatively smaller diameter (e.g. 0.08 xcexcm) are then added. These smaller particles are coated with rabbit anti-IgE antibodies and if larger particles have IgE bound to the allergen these small particles will bind to these antibodies and will form by virtue of agglomeration reaction so-called agglutination particles, i.e., large particles surrounded by several smaller particles. However, these particles are bound to each other via non-covalent binding and the agglomeration occurs as the consequence and result of the presence of the analyte of interest. No admission is made that any reference cited in this specification is prior art. All references cited herein are hereby expressly incorporated by reference.
This invention involves microparticles which are uniquely distinguished by detectable characteristics. In addition, the microparticles have bound to them reagents which react with analytes in samples to be analyzed in bimolecular reactions. The results of the reactions are bound to the microparticles and are measured, thereby allowing quantification of the amount of analyte in each sample. A variety of instruments are used to characterize the microparticles and simultaneously measure the bimolecular reactions. These include flow cytometry, electrophoresis cells, and centrifuges.
This invention provides significant advantages and efficiencies in the analysis of clinical and other samples from a single source for a number of analytes, and for analyzing a number of samples from a variety of sources for a single analyte. Any system or instrument capable of separating microparticles into subpopulations according to specified characteristics and of determining a chemical reaction using the same detection method as for the characteristics may be used with this invention. For example, flow cytometry using fluorescence detection is the embodiment described in greatest detail below. Other suitable systems include free flow electrophoresis and centrifugation, both of which may use detection means based on fluorescence, electrical charge, impedance, magnetic properties, etc.
Accordingly, this invention provides a novel article, which comprises a polymer microparticle having attached to its surface one or more populations or sets of fluorescently stained nanoparticles. All nanospheres in a given population are dyed with the same concentration of a dye, and by coupling a predetermined number of these nanospheres to the microparticle, along with known quantities of other nanospheres stained with different dyes, a multifluorescent microsphere results. By varying the quantity and ratio of different populations or sets of nanospheres it is possible to establish and distinguish a large number of discreet populations of carrier particles with unique emission spectra or fluorescence signal.
It is an object of the invention to provide a novel article, which comprises a microparticle carrying on its surface one or more populations of fluorescently stained nanoparticles. All nanospheres in a given population are dyed with the same concentration of a dye, and by coupling a known quantity of these nanospheres to the microparticle, along with known quantities of other nanospheres stained with different dyes, a multifluorescent microsphere results. By varying the quantity and ratio of different populations of nanospheres it is possible to establish and distinguish a large number of discreet populations of carrier particles with unique emission spectra. The carrier particles can be stained as well to provide an additional color or signal.
Although the article of the invention could appear as being similar to existing agglutination particles disclosed by Masson et al., and other similar prior art disclosures, they are patentably distinct from the instant invention, because means and the sequential order of particle-to-particle coupling are radically dissimilar. The purpose of the agglutination assay and means of the detection are also drastically different. Thus the prior art disclosures relating to particle agglutination methods and compositions are totally irrelevant and unrelated to the instant invention. The article of the instant invention requires that it is formed prior to the detection of an analyte of interest.
Polymeric microparticles used in this invention as carrier or core particles have a diameter of less than one millimeter, preferably having a size ranging from about 0.1 to about 1,000 micrometers (xcexcm) in diameter. Even though the microparticle can be of any size, the preferred size is 1-100 xcexcm, more preferably 2-50 xcexcm, more preferably 3-25 xcexcm, and even more preferably about 6-12 xcexcm.
Preferred sizes for nanoparticles range from about 1 nanometer (nm) to about 100,000 nm in diameter. Optimally preferred diameters are within about 10 and 1,000 nm, preferably within 100 and 800 nm, and more preferably within 200 and 500 nm.
It is a further object of the invention to provide nanospheres as well as carrier particles, which are preferably made of polymer material, i.e., polystyrene. However, polymeric materials including but not limited to brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyamide, polyacrylamide, polyacrolein, polybutadiene, polycaprolactone, polycarbonate, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, polylactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, polyphosphazene, polyphosophaze, polysulfone, or combinations thereof are acceptable as well. Other polymer materials such as carbohydrate, e.g., carboxymethyl cellulose, hydroxyethyl cellulose, agar, gel, proteinaceous polymer, polypeptide, eukaryotic and prokaryotic cells, viruses, lipid, metal, resin, latex, rubber, silicone, e.g., polydimethyldiphenyl siloxane, glass, ceramic, charcoal, kaolinite, bentonite, and the like can be equally used.
These polymers may also incorporate magnet or magnetically responsive metal oxides selected from the group consisting of superparamagnetic, paramagnetic, and ferromagnetic metal oxide.
It is a still further object of this invention to provide particles which will contain about 0% to 70% (weight/weight) of a cross-linking agent, such as divinyl benzene, ethylene glycol dimethacrylate, trimethylol propane trimethacrylate, or N,Nxe2x80x2methylene-bis-acrylamide or other functionally equivalent agents known in the art. In a preferred embodiment, core microspheres, as well as nanospheres, are made of polystyrene and contain about 0% to 30% divinyl benzene.
It is a still further object of this invention to provide particles which will contain additional surface functional groups such as carboxylates, esters, alcohols, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides, or halides, which may facilitate attachment of analytical reactants and/or particle-to-particle bonding.
It is a still further object of the invention to provide methods of preparing microparticles by covalent coupling or any other known means of coupling, e.g., ionic bonds, hydrogen bonds, or by simple adsorption. Other methods of coupling are provided, including, the coupling by an adsorption followed by surrounding the article of invention with a polymeric shell.
It is a still further object of the invention to provide dyes that are fluorescent dyes. Preferably such dyes are hydrophobic and are capable of staining polymeric particles. The preferred dyes are cyanine dyes. For specific purposes such as labeling label or detection reagents, i.e., an antibody, the hydrophilic dyes such as fluorescein (FITC) can be used.
It is a still further object of the invention to provide dyes with light emission at a wavelength in the ultra-violet or visible range. It is a still further object of the invention to provide dyes that fluoresce in the infrared or near infrared region.
It is a still further object of the invention to provide dyes with light emission at a wavelength of greater than about 450 nm, preferably greater than about 480 nm, more preferably at greater than about 500 nm. The preferred dyes are polymethine cyanines or squaraines, which emit fluorescent light having wavelengths in the region of about 500 nm to about 1000 nm. Preferably, when more than one dye is used to stain more than one population of nanospheres, these dyes are chosen such that they possess substantially different emission spectra, preferably having emission maxima separated by greater than 10 nm, more preferably having emission maxima separated by greater than 25 nm, even more preferably separated by greater than 50 nm. Dyes can be selected to have emission bands that match commercially available filters or for detecting multiple fluorophores with several excitation and emission bands.
It is a still further object of the invention to provide methods of using such particles for various diagnostic, analytic, and industrial applications known in the art. It is a still further object of the invention to provide a kit suitable for use in detection of the analytes of interest. Preferably, this kit contains a series of microparticles with attached nanoparticles having a distinct fluorescent signal and also an analytical reactant capable of specifically binding with one of analytes of interest. The kit also contains a secondary reagent comprising a reagent, which binds to the same analyte as the analytical reagent and also this kit may contain a fluorescent label, a competitor molecule, a reference material, and other ingredients that are accepted as standard reagents such as a wash buffer, necessary plasticware, etc.
It is a still further object of the invention to detect and analyze various analytes which can be in a broader sense an antigen, an antibody (both monoclonal and polyclonal), a receptor, a hapten, an enzyme, a protein, a peptide, a nucleic acid, a drug, a hormone, a chemical, a polymer, a pathogen, a toxin, or combination thereof.
It is a further object of this invention to provide methods for detecting multiple subpopulations of analytes of interest in a sample employing multicolored particles of the instant invention.
In accordance with the above and further objects of the invention, the preferred method to detect, differentiate, sort, quantitate, and/or analyze aspects or portions of analytes in a sample is by flow cytometry.
It is a further object of this invention to provide other means for detection and analysis, including, but not limited to visual inspection, digital (CCD) cameras, video cameras, photographic film, or the use of current instrumentation such as laser scanning devices, fluorometers, luminometers, photodiodes, quantum counters, plate readers, epifluorescence microscopes, scanning microscopes, confocal microscopes, capillary electrophoresis detectors, or by other means for amplifying the signal such as a photomultiplier tube or other light detector capable of detecting the presence, location, intensity, excitation and emission spectra, fluorescence polarization, fluorescence lifetime, and other physical properties of the fluorescent signal.
It is a further object of this invention to provide rapid and efficient methods for analyzing samples of several origins for a single analytes.
It is a further object of this invention to provide rapid and efficient methods for analyzing samples of a single origin for a variety of analytes.
Before the present invention is disclosed and described, it is to be understood that this invention is not limited to the particular process steps and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.
It must be noted that, as used in this specification and the appended claims, the singular forms xe2x80x9ca,xe2x80x9d xe2x80x9can,xe2x80x9d and xe2x80x9cthexe2x80x9d include plural references unless the content clearly dictates otherwise. Thus, for example, reference to a method of staining xe2x80x9ca particlexe2x80x9d with xe2x80x9ca dyexe2x80x9d may include a mixture of one or more dyes and one or more particles. As used hereinafter the terms fluorescent dye, fluorescer, fluorochrome, or fluorophore are used interchangeably and bear equivalent meanings.
Particles
As used hereinafter the terms microparticles, microspheres, or microbeads are used interchangeably and bear equivalent meanings as they refer to small particles with overall diameter that falls essentially in the micrometer range. The terms nanospheres, nanoparticles, or nanobeads refer to smaller particles with overall size that falls essentially in the nanometer range. As used hereinafter the general term particles, spheres, or beads refers both to microparticles and nanoparticles, which effectively serve as solid supports or solid phase.
Fluorescent polymer nanospheres coupled to carrier microparticles are described. Fluorescently distinguishable particle sets are obtained through variation of the amount and type of dye in the nanospheres. The desirable number of nanospheres linked to the microparticle, and ratios of differently dyed nanospheres on the surface of the carrier particle are selected according to particular need.
Nanospheres used in this invention are commercially available in sizes ranging from about 10 nanometers (nm) to about 100,000 nm in diameter. Optimally preferred diameters are within about 10 and 1,000 nm, preferably within 200 and 500 nm. Polymeric microspheres used in this invention as carrier particles to which nanospheres are bound normally range in size from 0.01 to 1000 micrometers (xcexcm) in diameter. Even though the microparticle can be of any size, the preferred size is 0.1-500 xcexcm, more preferably 1-200 xcexcm, and even more preferably 2-12 xcexcm. The particles can be uniform (being about the same size) or of variable size such that the differences can be determined by size-dependent properties such as light scattering or optical refraction.
Particles are made of any regularly shaped material. The preferred shape is spherical, however, particles of any other shape can be employed since this parameter is immaterial to the nature of the invention. The shape of the particle can serve as an additional distinction parameter, which is discriminated by flow cytometry, e.g., by a high-resolution slit-scanning method.
Usually these nanospheres as well as carrier particles are made of the same material such as polystyrene or latex. However, other polymeric materials are acceptable including polymers selected from the chemical group consisting of carbohydrate-based polymers, polyaliphatic alcohols, poly(vinyl) polymers, polyacrylic acids, polyorganic acids, polyamino acids, co-polymers, block co-polymers, tertpolymers, polyethers, naturally occurring polymers, polyimids, surfactants, polyesters, branched polymers, cyclo-polymers, polyaldehydes and mixtures thereof. More specifically, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyamide, polyacrylamide, polyacrolein, polybutadiene, polycaprolactone, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, polylactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, polyphosphazene, polyphosophaze, or combinations thereof are preferable. Representative combination polymers of which the polymeric particles are composed include for example poly-(styrene-co-vinylbenzyl chloride-co-acrylic acid) (85:10:5 molar ratio), poly(styrene-co-acrylic acid) (99:1 molar ratio), poly(styrene-co-methacrylic acid) (90:10 molar ratio), poly(styrene-co-acrylic acid-co-mandp-divinylbenzene) (89:10:1 molar ratio), poly-(styrene-co-2-carboxyethyl acrylate) (90:10 molar ratio), poly(methyl methacrylate-co-acrylic acid) (70:30 molar ratio) and poly(styrene-co-butyl acrylate-co-methacrylic acid)(45:45:10 weight ratio). Most of beads formed from synthetic polymers such as polystyrene, polyacrylamide, polyacrylate, or latex are now commercially available from numerous sources such as Bio-Rad Laboratories (Richmond, Calif.) and LKB Produkter (Stockholm, Sweden). Beads formed from natural macromolecules and particles such as agarose, crosslinked agarose, globulin, deoxyribose nucleic acid, and liposomes are commercially available from sources such as Bio-Rad Laboratories, Pharmacia (Piscataway, N.J.), and IBF (France). Beads formed from copolymers of polyacrylamide and agarose are commercially available from sources such as IBF and Pharmacia.
These polymers may also incorporate magnet or magnetically responsive metal oxide selected from the group consisting of superparamagnetic, paramagnetic, and ferromagnetic metal oxide. Magnetic beads are commercially available from sources such as Dynal Inc. (Great Neck, N.Y.) or can be prepared using known in the art methods as disclosed for example in U.S. Pat. Nos. 4,358,388; 4,654,267; 4,774,265; 5,320,944; and 5,356,713.
Other materials such as carbohydrate, e.g., carboxymethyl cellulose, hydroxyethyl cellulose, proteinaceous polymer, polypeptide, eukaryotic and prokaryotic cells, viruses, lipid, metal, resin, rubber, silica, silicone, e.g., polydimethyldiphenyl siloxane, glass, ceramic and the like can be equally used.
The nanoparticles are preferably made of the same material as the microparticles. However, if required, they can be made of different material. It is to be understood that in this Specification the terms xe2x80x9cfirstxe2x80x9d and xe2x80x9csecondxe2x80x9d, as applied to polymer species which compose microparticles and nanoparticles are used for the purposes of identification only and do not imply any order of preference.
The microspheres will also contain approximately 0% to 70% of a cross-linking agent, such as divinyl benzene, ethylene glycol dimethacrylate, trimethylol propane trimethacrylate, or N,Nxe2x80x2methylene-bis-acrylamide or other functionally equivalent agents known in the art. Crosslinking of carbohydrate polymer such as hydroxypropyl cellulose can be achieved with adipic acid, sebacic acid, succinic acid, citric acid, 1,2,3,4-butanetetracarboxylic acid, or 1,10 decanedicarboxylic acid. In a preferred embodiment, core microspheres and nanospheres are made of polystyrene and contain about 0% to 30% divinyl benzene.
The particles may have additional surface functional groups to facilitate the attachment and bonding. These groups may include carboxylates, esters, alcohols, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides, or halides. Carboxylated latex particles have been used to prepare diagnostic reagents as described, for example, in U.S. Pat. No. 4,181,636. As described therein, the conventional procedure for covalently attaching an immunologically reactive species to the particles having surface carboxyl groups involves the use of a water-soluble carbodiimide. For many practical applications it is critical that the polymeric particles have surface carboxyl groups available for attachment of the reactive amine- or sulfhydryl-containing compound. Such groups are preferably added to the particles by incorporating monomers containing such groups into the polymers (for example, acrylic acid, methacrylic acid, itaconic acid, and the like). Alternatively, they can be added to the particles by further chemical reaction of a polymer having other precursor reactive groups which can be converted to carboxyl groups (for example, by hydrolysis of anhydrides, such as maleic anhydride, or by oxidation of surface methylol or aldehyde end groups). Other compounds, such as diamines, dihydrazides, mercaptoalkylamines and dimercaptans can be used as linking moieties for later attachment of drugs, enzymes or other reactive species such as nanospheres. Although the preferred attaching or bonding method is by covalent linkage, other methods such as adsorption can be equally used. Other novel methods such as surrounding microparticle-nanoparticle complexes by a polymeric shell are acceptable as well.
Dyes
Fluorescent dyes used in this invention are preferebly of the general class known as cyanine dyes, with emission wavelengths between 550 nm and 900 nm. These dyes may contain methine groups and their number influences the spectral properties of the dye. The monomethine dyes that are pyridines typically have blue to blue-green fluorescence emission, while quinolines have green to yellow-green fluorescence emission. The trimethine dye analogs are substantially shifted toward red wavelengths, and the pentamethine dyes are shifted even further, often exhibiting infrared fluorescence emission (see,for example, U.S. Pat. No. 5,760,201).
However, any dye that is soluble in an organic solvent can be used. The squaric acid based fluorescent dyes can be synthesized by methods described in the literature. See, for example, Sprenger et al. Angew. Chem., 79, 581 (1967); Angew. Chem., 80, 541 (1968); and Maaks et al., Angew Chem. Intern. Edit., 5, 888 (1966). Additionally, unsymmetrically substituted squaric acid compounds can be synthesized by methods such as those described by Law et al., J. Org. Chem. 57, 3278, (1992). Specific methods of making some of such dyes are well known in the art and can be found for example in U.S. Pat. Nos. 5,795,981; 5,656,750; 5,492,795; 4,677,045; 5,237,498; and 5,354,873. The practical use of above the described fluorescent dyes, e.g., phthalocyanines, 2,3-naphthalocyanines, squaraines and croconic acid derivatives is disclosed in U.S. Pat. No. 5,525,516 issued to Krutak, et al.
In addition to fluorescent dyes used in this preferred embodiment, related dyes can be further selected from cyclobutenedione derivatives, substituted cephalosporin compounds, fluorinated squaraine compositions, symmetrical and unsymmetrical squaraines, alkylalkoxy squaraines, or squarylium compounds. Some of these dyes can fluoresce at near infrared as well as at infrared wavelengths that would effectively expand the range of emission spectra up to about 1,000 nm. In addition to squaraines, i.e., derived from squaric acid, hydrophobic dyes such as phthalocyanines and naphthalocyanines can also be selected to operate at longer wavelengths. Other classes of fluorochromes are equally suitable for use as dyes according to the present invention. Some of these dyes are listed herein: 3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine, 5-Hydroxy Tryptamine (5-HT), Acid Fuhsin, Acridine Orange, Acridine Red, Acridine Yellow, Acriflavin, AFA (Acriflavin Feulgen SITSA), Alizarin Complexon, Alizarin Red, Allophycocyanin, ACMA, Aminoactinomycin D, Aminocoumarin, Anthroyl Stearate, Aryl- or Heteroaryl-substituted Polyolefin, Astrazon Brilliant Red 4 G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine Sulphate, Bisbenzamide, BOBO 1, Blancophor FFG Solution, Blancophor SV, Bodipy F1, BOPRO 1, Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green, Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution, Calcophor White Standard Solution, Carbocyanine, Carbostyryl, Cascade Blue, Cascade Yellow, Catecholamine, Chinacrine, Coriphosphine O, Coumarin, Coumarin-Phalloidin, CY3.1 8, CY5.1 8, CY7, Dans (1-Dimethyl Amino Naphaline 5 Sulphonic Acid), Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH-CH3, DAPI, Diamino Phenyl Oxydiazole (DAO), Dimethylamino-5-Sulphonic acid, Dipyrrometheneboron Difluoride, Diphenyl Brilliant Flavine 7GFF, Dopamine, Eosin, Erythrosin ITC, Ethidium Bromide, Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange, Fluo 3, Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid, Granular Blue, Haematoporphyrin, Hoechst 33258, Indo-1, Intrawhite Cf Liquid, Leucophor PAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200), Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (Methyl Green Pyronine Stilbene), Mithramycin, NBD Amine, Nile Red, Nitrobenzoxadidole, Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan Brilliant Flavin E8G, Oregon Green, Oxazine, Oxazole, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), Phorwite AR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R, Phthalocyanine, Phycoerythrin R, Polyazaindacene Pontochrome Blue Black, Porphyrin, Primuline, Procion Yellow, Propidium Iodide, Pyronine, Pyronine B, Pyrozal Brilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5 GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra, Rhodamine BB, Rhodamine BG, Rhodamine WT, Rose Bengal, Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, SITS (Primuline), SITS (Stilbene Isothiosulphonic acid), Stilbene, Snarf 1, sulpho Rhodamine B Can C, Sulpho Rhodamine G Extra, Tetracycline, Texas Red, Thiazine Red R, Thioflavin S, Thioflavin TCN, Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, TOTO 1, TOTO 3, True Blue, Ultralite, Uranine B, Uvitex SFC, Xylene Orange, XRITC, YO PRO 1, or combinations thereof. Optionally such dyes will contain functional groups capable of forming a stable fluorescent product with functional groups typically found in biomolecules or polymers including activated esters, isothiocyanates, amines, hydrazines, halides, acids, azides, maleimides, alcohols, acrylamides, haloacetamides, phenols, thiols, acids, aldehydes and ketones.
One skilled in the art would know which one to select among such dyes as long as the desired emission and absorption properties as well as their hydrophobic properties are appropriate. The spectral properties of the fluorescent dyes should be sufficiently similar in excitation wavelengths and intensity to fluorescein or rhodamine derivatives as to permit the use of the same flow cytometry equipment. It is preferable that the dyes, however, have higher solubility in organic solvents and have improved photostability and quantum yields.
More preferably, the dyes have the same or overlapping excitation spectra, but possess distinguishable emission spectra. Any detection system can be used to detect the difference in spectral characteristics between the two dyes, including a solid state detector, photomultiplier tube, photographic film, or eye, any of which may be used in conjunction with additional instrumentation such as a spectrometer, luminometer microscope, plate reader, fluorescent scanner, flow cytometer, or any combination thereof, to complete the detection system. Preferably dyes are chosen such that they possess substantially different emission spectra, preferably having emission maxima separated by greater than 10 nm, more preferably having emission maxima separated by greater than 25 nm, even more preferably separated by greater than 50 nm. When differentiation between the two dyes is accomplished by visual inspection, the two dyes preferably have emission wavelengths of perceptibly different colors to enhance visual discrimination. When it is desirable to differentiate between the two dyes using instrumental methods, a variety of filters and diffraction gratings allow the respective emission maxima to be independently detected. When two dyes are selected that possess similar emission maxima, instrumental discrimination can be enhanced by insuring that both dyes"" emission spectra have similar integrated amplitudes, similar bandwidths, and the instrumental system""s optical throughput be equivalent across the emission range of the two dyes. Instrumental discrimination can also be enhanced by selecting dyes with narrow bandwidths rather than broad bandwidths, however such dyes must necessarily possess a high amplitude emission or be present in sufficient concentration that the loss of integrated signal strength is not detrimental to signal detection.
Staining Process
The technology is available enabling one skilled in the art to make a series of multicolored, fluorescent particles with unique fluorescence characteristics and using such particles for multiparameter analysis of a plurality of analytes. According to this technology, both microparticles and nanoparticles can be subjected to fluorescent staining with distinct dyes. In the preferred embodiment, the nanoparticles are stained and more preferably more than one set of nanoparticles are provided which are stained with one or more distinct fluorescent dyes. Fluorescent staining of polymeric particles may be achieved by any of the techniques familiar to those skilled in the art. Three distinct means of making fluorescent particles are known, including: (i) covalent attachment of dyes onto the surface of the particle, (ii) internal incorporation of dyes during particle polymerization, and (iii) dyeing after the particle has already been polymerized.
(i) U.S. Pat. Nos. 5,194,300 Cheung and 4,774,189 Schwartz, disclose, for example, fluorescent microspheres that are coated by covalently attaching to their surface one or more fluorescent dyes.
(ii) U.S. Pat. Nos. 5,073,498 Schwartz and 4,717,655 Fulwyler, disclose fluorescent dyes added during particle polymerization process.
(iii) The principle of the third method, i.e., internally embedding or diffusing a dye after a particle has been already polymerized was originally described by L.B.Bangs (Uniform Latex Particles; Seragen Diagnostics Inc. 1984, p. 40). U.S. Pat. No. 5,723,218 issued to Haugland et al. discloses diffusely dyeing microparticles with one or more dipyrrometheneboron difluoride dyes by using a process, which is essentially similar to the Bangs method. The combination of above methods are also possible and these examples are offered by way of illustration and not by way of limitation. One such technique is described here:
A 1% w/w solution of nanospheres (300 nm diameter polystyrene, amino functionalized) is stirred in a round bottom flask. To this is added a solution of the dye in an organic solvent, such as chloroform. When the dye solution is no longer absorbed by the nanospheres, addition of the dye is halted and the solvent is removed under reduced pressure.
In another embodiment, two fluorescent dyes are used, e.g., red squaraine dye which is 1,3-bis [(1,3-dihydro- 1,3,3-trimethyl-2H-indol-2-ylidene)methyl]-2,4-dihydroxy-cyclobutenediylium, bis(inner salt) and orange squaraine dye is 2-(3,5-dimethylpyrrol-2-yl)-4-(3,5-dimethyl-2H-pyrrol-2-ylidene)-3-hydroxy- 2-cyclobuten 1-one. These dyes are used to stain two separate populations of nanospheres. Alternatively, one population of nanospheres and carrier microsphere are stained. As another alternative, two dyes are combined at different ratio within the same particle of a given population.
Optimal staining with a particular dye is dependent upon the physical and chemical nature of the individual dye or polymeric substrate and the dye medium, as well as the property being assessed. Incubation times may vary widely depending on the desirable results, the concentration of the dye and the particles and the reaction conditions. The optimal time is usually the minimum time required for the dye, in the concentration being used, to achieve the highest specific signal while avoiding degradation of the dye over time and minimizing all other undesirable fluorescent signals due to the dye.
The total dye quantity is between about 0.00001% and 15% by weight to particle weight. This limitation, is however, of little consequence to the present invention for as long as the particle impregnated with said dyes is stable and usable for its intended purpose.
Both dyes would preferably be excited at the same absorption wavelength, e.g., ranging from ultraviolet to about 800 nm, and emit fluorescent light at two distinct, essentially non-overlapping wavelengths distant from each other by at least 10 nm, preferably 30 nm, and more preferably by at least 50 nm. For example, the emission peak of the first dye may be at 585 nm, and the peak emission of the second dye may be at 630 nm.
Alternatively, the dye of the invention is selected to give a detectable response that is different from that of other reagents desired to be used in combination with the subject dyes. For example, dyes that form complexes that permit excitation beyond 600 nm can be used in combination with commonly used fluorescent antibodies such as those labeled with fluorescein isothiocyanate or phycoerythrin.
Any fluorescence detection system (including visual inspection) can be used to detect differences in spectral properties between dyes, with differing levels of sensitivity. Such differences include, but are not limited to, a difference in excitation maxima, a difference in emission maxima, a difference in fluorescence lifetimes, a difference in fluorescence emission intensity at the same excitation wavelength or at a different wavelength, a difference in absorptivity, a difference in fluorescence polarization, a difference in fluorescence enhancement in combination with target materials, or combinations thereof. The detectably different dye is optionally one of the dyes of the invention having different spectral properties and different selectivity. In one aspect of the invention, the dye-particle complex and the additional detection reagents have the same or overlapping excitation spectra, but possess visibly different emission spectra, generally having emission maxima separated by  greater than 10 nm, preferably  greater than 20 nm, more preferably  greater than 50 nm. Simultaneous excitation of all fluorescent reagents may require excitation of the sample at a wavelength that is suboptimal for each reagent individually, but optimal for the combination of reagents. Alternatively, the additional reagent(s) can be simultaneously or sequentially excited at a wavelength that is different from that used to excite the subject dye. In yet another alternative, one or more additional reagents are used to quench or partially quench dye emission.
In a preferred embodiment, chlorinated solvents, more preferably chloroform, are used to solubilize dyes. However, suitable solvents are selected based on their ability to solubilize the particular class of hydrophobic dyes of interest. The solvents can be acyl, aliphatic, cycloaliphatic, aromatic or heterocyclic hydrocarbons; the solvents may or may not have halogens, oxygen, sulfur, nitrogen, and/or phosphorus as either terminal groups or as integral parts of a ring or chain. Specifically, solvents such as alcohol, ethyl acetate, toluene, xylene, hexane, pentane, benzene, ether, acetone, oil, carbone tetrachloride, carbon disulfide, DMSO, or methylene chloride can be used. Other solvents known in the art can be used and may be selected among various solvents listed in the Merck Index (Eleventh Edition, see section MISC-63-68).
The resulting dyed nanospheres may then be linked to the dyed or undyed microparticles by any of the well-known coupling reactions such as carbodiimide coupling (see below). Other methods of coupling using carboxylates, esters, alcohols, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides, or halides can be used as well by methods well known in the art.
Methods of Using the Article of Invention
The present invention provides a fluorescent polymeric article, comprising a carrier microparticle carrying one or more nanoparticles with multiple fluorescent signals. Optionally, the carrier microparticle of the invention can be also stained with a distinct fluorescent dye. To obtain such an article, a carrier particle is coated (covalently or by adsorption) with a plurality of smaller fluorescent particles (nanoparticles) with an average size from about 100 to 500 nanometers in diameter regardless of shape and composition. The article can be further coated or surrounded by a thin polymeric shell, selected in such a way that it would not affect light absorption and emission characteristics. This is accomplished by either of two separate methods described hereinafter.
The fluorescent nanoparticles of this invention may be prepared by incorporating either a single fluorescent dye and combining one or more populations of nanoparticles at different ratios. Alternatively, a combination of two or more fluorescent dyes within the same particle can be used to obtain multifluorescent nanoparticles. The fluorescence intensity of these fluorescent nanoparticles can be adjusted by varying the amount of fluorescent dye incorporated. The surface of both types of particles can be modified further to provide the desired surface characteristics allowing attachment of functional groups or chemical bonds.
In addition to flow cytometry, calibration purposes the fluorescent particles having these characteristics are useful in a wide variety of biomedical applications. The analytical method is also provided which is based on using multicolored fluorescent microparticles obtained by the instant invention. When each such population of microparticles, characterized by at least two fluorescent signals, is combined with an analytical reactant capable of binding a specific analyte of interest in a clinical or test sample a powerful analytical tool is obtained, which can provide qualitative and quantitative assay results. To achieve truly multiplexed analysis of a plurality of analytes in a sample, a third type of fluorescent signal, e.g., green fluorescent signal (FITC) is provided, usually found in a label reagent, which is capable of binding the analyte of interest. The label reagent as defined herein thus has two functions: the capacity to react with the analyte and the capacity to provide a fluorescent signal, which is distinct from the fluorescence signal of the particle of the invention. An ordinary flow cytometer is capable of analyzing spectral properties (fluorescent signals) of up to 20,000 particles per second and can provide reliable quantitative data in real-time scale. Thus, methods of making multicolored microparticles, the microparticles themselves, multiple sets of such microparticles, and multiplexed methods of analyzing a plurality of analytes in a sample are claimed by the instant invention.
A suitable flow cytometer for use with this invention is the Coulter Elite-ESP flow cytometer, or FACScan flow cytometer available from Beckman Coulter, Inc., Fullerton, Calif. Also suitable is the MOFLO flow cytometer available from Cytomation, Inc., Fort Collins, Colo.
In addition to flow cytometry, a centrifuge may be used as the instrument to separate and classify the microparticles. A suitable system is that described in U.S. Pat. No. 5,926,387, incorporated herein by reference.
In addition to flow cytometry and centrifugation, a free-flow electrophoresis apparatus may be used as the instrument to separate and classify the microparticles. A suitable system is that described in U.S. Pat. No. 4,310,408, incorporated herein by reference.
The fluorescent article of the invention can be used for passive or covalent coupling of biological material, i.e., analyte or analytical reactant, such as haptens, antigens, antibodies, enzymes or nucleic acids and used for various types of analyte assays such as immunoassays, nucleic acid (DNA or RNA) assays, affinity purification, cell separation and other medical, diagnostic, and industrial applications.
A large number of protocols exist for detecting the various analytes of interest including proteins of one hundred or more amino acids, peptides of less than one hundred amino acids, polysaccharides, nucleic acids, organic drugs, inorganic drugs, cells, and tissues. The protocols may involve use of a signal producing system, which involves a labeled conjugate, which may be directly or indirectly detected. These techniques may employ dyes, enzymes, enzyme substrates or co-factors, enzyme inhibitors, fluorescers, chemiluminescers, particles, or the like.
Analyte and analyte reactant pairs may, for example, be selected from any of the following combinations, in which either member of the pair may be the analyte and the other the binding partner, e.g., antigen and specific antibody; hormone and hormone receptor; hapten and anti-hapten; polynucleotide and complementary polynucleotide; polynucleotide and polynucleotide binding protein; biotin and avidin or streptavidin; enzyme and enzyme cofactor; and lectin and specific carbohydrate. The term analyte as used hereinafter is a compound or composition to be measured, which is mono- or polyvalent, that is, having one or a plurality of determinant sites, haptenic and antigenic, a single compound or plurality of compounds which share at least one common epitopic or determinant site; or a receptor. The term receptor as used hereinafter is any macromolecular compound or composition capable of recognizing (having an enhanced binding affinity to) a particular spatial and polar organization of a molecule, i.e., epitopic or determinant site. Illustrative receptors include naturally occurring receptors, e.g., thyroxine binding globulin, antibodies, enzymes, immunoglobulin (Fab) fragments, lectins, various proteins found on the surface of cells (cluster of differentiation or CD molecules), and the like. CD molecules denote known and unknown proteins on the surface of eukaryotic cells, e.g., CD4 is the molecule that primarily defines helper T lymphocytes. The term antibody is employed in this case as illustrative of, and to more generally denote receptor. In turn the term ligand is any compound or substance for which a receptor naturally exists or can be prepared.
The haptens may include naturally occurring hormones, naturally occurring drugs, synthetic drugs, pollutants, allergens, affector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, oligopeptides, chemical intermediates, nucleotides, oligonucleotides or the like. The use for such compounds may be in the detection of drugs of abuse, therapeutic dosage monitoring, health status, donor matching for transplantation purposes, pregnancy (e.g., hCG or alpha-fetoprotein), detection of disease, e.g. endotoxins, cancer antigens, pathogens, and the like. Therapeutic drugs may include, but are not limited to, anti-AIDS substances, anti-cancer substances, antibiotics, anti-viral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson substances, anti-spasmotics and muscle contractants, miotics and anti-cholinergics, iminunosuppressants (e.g. cyclosporine) anti-glaucoma solutes, anti-parasite and/or anti-protozoal solutes, anti-hypertensives, analgesics, anti-pyretics and anti-inflammatory agents (such as NSAID""s), local anesthetics, ophthalmics, prostaglandins, anti-depressants, anti-psychotic substances, anti-emetics, imaging agents, specific targeting agents, neurotransmitters, proteins and cell response modifiers. Proteins are of interest in a wide variety of diagnostics, such as detecting cell populations, blood type, pathogens, immune responses to pathogens, immune complexes, saccharides, lectins, naturally occurring receptors, and the like. Receptors may find use in binding to haptens, proteins, other receptors, or the like, or detection of the presence of pathogens, the level of a particular protein in a physiological fluid, the presence of haptens in a wide variety of samples, such as physiological fluids, air, process streams, water, etc. Nucleic acids may also find use in the detection of complementary strands, proteins specifically binding to nucleic acids, and the like.
The analytical reactants can be also selected among fluorescent reporter molecules capable to react with a variety of inorganic analytes that define properties of biological fluids, air, water, and the like, e.g., O2, CO2, pH, Ca++, Na+, K+, or Clxe2x88x92 as disclosed for example in U.S. Pat. No. 5,747,349 issued to van den Engh et al.
Of particular interest is the binding of microorganisms and cells, including viruses, prokaryotic and eukaryotic cells, unicellular and polycellular organism cells, e.g., fungi, animal, mammal, etc., or fragments thereof. Usually, these large aggregations will be non-covalently bound to the surface through specific binding pair member complexes. By having a high density of binding members bound to the surface, a cell or virus may be complexed by a large number of binding pair members, providing very strong anchoring of the cell, virus, or fragment. The system may then be subjected to vigorous treatment without concern for dislodging the specifically bound entity, while non-specifically bound materials may be readily removed.
The subject of the invention may also be used for detecting pathogens. Monoclonal antibodies may be linked to the surface to serve as catching antibodies. The sample would then be added and cells having the epitope recognized by the antibody would bind to the antibody on the surface. Non-specifically bound pathogens are washed away leaving substantially only specifically bound ones. Labeled monoclonal antibodies are then added which are specific for an epitope other than the epitope recognized by the catching antibody. The term epitope is synonymous to term antigenic determinant and as used herein means a defined domain on the molecule that serves as a reaction or binding site. A molecule may have more than one epitope. For example, first epitope would allow coupling of the analyte with respective analytical reactant and second epitope will provide a binding site or domain for the label reagent. In contrast, a competitor molecule will be interfering (competing) with the formation of a binding pair analyte-analytical reactant. After incubating to allow reaction between the antibodies and pathogens, non-specifically bound antibodies are washed away and the presence of the label determined according to standard detection methods. Pathogens of interest may be viruses such as Herpesviruses, Poxviruses, Togaviruses, Flaviviruses, Picornaviruses, Orthomyxoviruses, Paramyxoviruses, Rhabdoviruses, Coronaviruses, Arenaviruses, and Retroviruses. They may also include bacteria including but not limited to Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Salmonella typhimurium, Staphylococcus epidermidis, Serratia marcescens, Mycobacterium bovis, methicillin resistant Staphylococcus aureus and Proteus vulgaris. The examples of such pathogens are not limited to above pathogens and one skilled in the art will know which specific species of microoragnisms and parasites are of particular importance. The non-exhaustive list of these organisms and associated diseases can be found for example in U.S. Pat. No. 5,795,158 issued to Warinner and incorporated herein by reference.
For detection or quantitation of a target molecule of interest or analyte, a sample is combined with a solution containing the microparticles, the macromolecules on the microparticles are reacted with the analyte, the microparticles are separated from any non-bound components of the sample, and microparticles containing bound molecules are detected by conventional methods. Fluorescently stained microparticles are particularly well suited for flow cytometry analysis in accordance with methods well known to those skilled in the art.
Coating of carrier particles for use in the method of the invention can be effected using, for example, procedures standard in the art. Thus, for example, representative techniques for coating particle systems with antibodies for use in immunoassay procedures are described by Frengen et al. in Clin. Chem. 39 (1993), pp. 2174-2181 and the references contained therein, and by Lindmo et al. in J. Immunol. Methods 126 (1990), pp. 183-189.
Assays using particles of the invention can be carried out in a biological fluid, including separated or unfiltered biological fluids such as urine, cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, amniotic fluid, gastric fluid, blood, serum, plasma, lymph fluid, interstitial fluid, tissue homogenate, cell extracts, saliva, sputum, stool, physiological secretions, tears, mucus, sweat, milk, semen, seminal fluid, vaginal secretions, fluid from ulcers and other surface eruptions, blisters, and abscesses, and extracts of tissues including biopsies of normal, malignant, and suspect tissues or any other constituents of the body which may contain the analyte of interest. Other similar specimens such as cell or tissue culture or culture broth are also of interest. Alternatively, the sample is obtained from an environmental source such as soil, water, or air; or from an industrial source such as taken from a waste stream, a water source, a supply line, or a production lot. Industrial sources also include fermentation media, such as from a biological reactor or food fermentation process such as brewing; or foodstuff, such as meat, game, produce, or dairy products. The test sample can be pre-treated prior to use, such as preparing plasma from blood, diluting viscous fluids, or the like; methods of treatment can involve filtration, distillation, concentration, inactivation of interfering compounds, and the addition of reagents.
A method for detecting multiple subpopulations of analytes of interest in a sample employing a complementary binding moiety to each of said analytes bound to a solid support, wherein each analyte and its complementary binding moiety comprise first and second members of a specific binding pair respectively is provided. The method includes the steps of forming a mixture of known proportions of multiple subpopulations of said complementary binding moieties, wherein each subpopulation comprises a different complementary binding moieties, contacting the sample with the mixture so that specific binding pairs are formed on the solid supports, and relating the presence of analytes of interest in the sample (U.S. Pat. No. 5,567,627 to Lehnen).
For the purposes of the present invention, the label or detectable fluorescent reagent should provide a signal related to the presence of analyte in the sample which results in the detection of electromagnetic radiation, particularly light in the ultra-violet, visible or infrared range.
Assays can be carried out in accordance with the various protocols. In accordance with the subject invention, the sample is contacted with the subject solid substrate and various operations may be carried out, such as the addition of miscellaneous reagents, incubations, washings, and the like. The final result of the assays will be the change in the amount of a product, which absorbs or produces light, either by light absorption or by light emission in relation to the presence or amount of the analyte of interest. Usually, this is as a result of formation of a specific binding complex between complementary members of a specific binding pair, where one of the members may serve as a bridge to form a sandwich (as in xe2x80x9csandwichxe2x80x9d assay), or there may be a single complex, or complexes may be bound to complex binding proteins, such as S. aureus protein A, rheumatoid factor, immunoglobulins specific for immune complexes, or the like.
By having fluorescent markers, such as fluorescent particles, fluorescent conjugated antibodies, or the like, the sample may be irradiated with light absorbed by the fluorescers and the emitted light measured by light measuring devices. Dyes can be employed as the label or produced as a result of a reaction, e.g. an enzymatically catalyzed reaction.
Similarly, with nucleic acid assays involving hybridization, one can carry out the necessary steps to determine whether complementary sequences are present, and by employing a wide variety of protocols, provide for a colored or fluorescent label or product of the label, which will indicate the presence or absence of the complementary sequence.
For example, one could activate the surface immediately prior to carrying out the assay by diazotizing the amino functionalities, add the nucleic acid sample to the activated surface, so as to be covalently bound, and then employ probes having a sequence complementary to the sequence of interest and functionalized, for example, by having a biotin label. After completion of the hybridization step, one could add enzyme or fluorochrome conjugated to avidin, which would bind to any biotin bound to the surface through hybridization. After washing away non-specifically bound avidin, the fluorochrome can be measured directly or the substrate for the enzyme could be added and the formation of product would be indicative of the presence and amount of the complementary sequence.
A variation would be to employ an antigen recognized by a cell receptor. The antigen would be bound to the surface to catch the cells and a labeled antigen used to label the cells. The receptor could be surface immunoglobulin. In this way the presence of the specifically bound cells could be determined, whereby having the antigen of interest complementary to the receptor bound to the surface, cells having the specific immunoglobulin for such antigen could be determined. Instead of having antigen, one would have antibodies to the antigen bound to the surface to non-covalently bind the antigen to the surface.
The subject article may also find use in isolating various products of interest, such as blood plasma proteins, growth factors, clotting factors, anti-clotting factors, or the like, which may then be released from the complex by various salt solutions. The article of the invention may be used for a variety of other purposes, whenever one wishes to provide a high density of oriented molecules at a surface or visualize events or provide for ready transmission of light, where the analyte substance is non-diffusively bound to a solid surface.